The p53 tumor suppressor is a homo-tetrameric, sequence-specific transcription factor that has crucial roles in apoptosis, cell cycle arrest, DNA repair, cellular senescence, metabolism and tumor suppression. It is maintained at low levels in unstressed cells but becomes stabilized and activated following DNA damage through extensive post-translational modification (PTM). Our research has focused on identifying and exploring the biological roles of p53 PTMs to better understand how they modulate p53 functions. Previously, we used structural and biophysical methods to characterize the complexes formed between the Taz2 domain of the transcriptional co-activator p300 and either the first (TAD1, residues 1-40) or second (TAD2, residues 35-59) transactivation subdomains of p53. Our results showed that both TAD1 and TAD2 subdomains occupy the same region of Taz2, form short alpha helices when bound, have similar affinities for Taz2, and are stabilized by both hydrophobic and electrostatic interactions. Although both TAD1 and TAD2 subdomains bind to p300, they also interact with distinct proteins and can function independently of one another. These observations suggest the existence of distinguishing transcriptional cofactors for TAD1 and TAD2 whose interaction is regulated differently by p53 phosphorylation. Comparison of the structures of the two complexes also suggests that these two p53 subdomains may function differently in co-activator recruitment after stress. In order to identify distinct interacting partners for TAD1 and TAD2, peptides comprising TAD1 (residues 9-33) or TAD2 (residues 35-59), with and without phosphorylation at Thr 18 or Ser 46, respectively, were synthesized and covalently attached to biotin at the N-termini. We used these peptides as bait for pulldown of interacting proteins from nuclear extracts prepared from MCF7 cells treated with etoposide. We used reductive dimethylation of peptides followed by mass spectrometry analysis to identify and quantitatively compare the interactors to discriminate between those preferentially interacting with the TAD1 and TAD2 subdomains. In our initial experiments using biological triplicate pulldowns, we identified several new potential interactors of p53 TAD1 and TAD2 in extracts from untreated or etoposide-treated cells that preferentially bind unmodified or modified p53 peptides. In addition, we are interested in the effects of PTMs in the p53 CTRD. The C-terminus of p53 is subject to diverse post-translational modifications, including phosphorylation, methylation, acetylation, ubiquitination, sumoylation, neddylation and hydroxylation, that are primarily localized to the terminal thirty residues of the protein. Previously, we have reported that p53 can be either mono- or dimethylated on Lys382, with the former modification repressing p53 transcriptional activity and the latter promoting DNA repair, in competition with demonstrated acetylation and ubiquitination of the same site. SETD8 monomethylates p53 on lysine 382, attenuating p53 pro-apoptotic and growth arrest functions. Using a high-content imaging siRNA screen and a chemical screen, in a collaboration with Drs. Veschi and Thiele, we identified SETD8 as a suppressor of p53 activity in neuroblastoma cell lines. Genetic or pharmacological inhibition of SETD8 activity resulted in activation of the p53 wild-type pathway by decreasing p53K382me1. Recently, we also demonstrated that inhibition of SETD8 overexpression in other types of cancer results in the activation of p53. Therefore, we are developing a high-throughput method to identify small molecule inhibitors of SETD8 with a lower IC50 and high tolerability in vivo. We will utilize a Rapid Fire 365 MS System in an activity-based high-throughput screen. Inhibition of SETD8 is a viable therapeutic strategy for tumors that have functionally inactivated wild-type p53. Finally, p53 point mutations have been reported to occur in approximately half of all tumors, with a marked over-representation of specific hot-spot residues. These mutations abolish the ability of p53 to function as a transcription factor or suppress tumor growth. Moreover, many mutant forms of p53 have novel oncogenic activities due to gain-of-function. Many hot-spot p53 mutants are structurally unstable under physiological conditions and form aggregates similar to those seen in amyloid diseases, thus resulting in protein inactivation. The development of effective inhibitors of mutant p53 protein aggregation requires the ability to determine the state of aggregation of the p53 protein in cells. In a recent Nature Communications paper (https://www.nature.com/articles/s41467-018-03599-w), a known deubiquitinase inhibitor, PR-619, was shown to specifically target p53-R175H for degradation. PR-619 disrupts the interaction between p53-R175H and the deubiquitinase, USP15, leading to increased ubiquitination and degradation. Our aim is to utilize the high-throughput, high-resolution Opera imaging system to screen libraries of small molecule and/or peptide mimetic compounds to identify suitable candidates that specifically disrupt the accumulation of mutant p53 and/or rescue p53 wildtype tumor suppressive function. We plan to use a variety of antibodies to probe the conformational state, localization, and total protein levels of p53 in cultured cells using the imaging system available to us at the National Center for Advancing Translational Sciences (NCATS).